Amplification of Nuclear Overhauser Effect by Hyperpolarization for Screening of Ligand Binding to Immobilized Target Proteins.,Analytical Chemistry

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Amplification of Nuclear Overhauser Effect by Hyperpolarization for Screening of Ligand Binding to Immobilized Target Proteins.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-09-08 , DOI: 10.1021/acs.analchem.0c01071
Yunyi Wang ₁ , Christian Hilty ₁

Affiliation

Immobilization of a target protein enhances the cross-relaxation rates for transfer of nuclear spin polarization but reduces the accessible target concentration. Hyperpolarization of ligand spins by dissolution dynamic nuclear polarization (D-DNP) is shown to increase sensitivity for observing the intraligand nuclear Overhauser effect (NOE). This effect, also known as the transferred NOE (trNOE), can be used for detection of binding and for obtaining binding-related structural information. The measurement of hyperpolarized trNOE signals is demonstrated for the ligand 4′-hydroxyazobenzene-2-carboxylic acid interacting with avidin protein immobilized on polystyrene beads. In a sample containing 63.5 μM ligands and 0.83 μM accessible protein binding sites, the signal enhancement provided by D-DNP leads to single-scan detection of the NOE buildup, despite that this signal peaks at only 2% of the total ligand signal. These buildup curves allow the confirmation of binding through a change in the sign of the NOE and the quantitative determination of cross-relaxation rates. The combination of the D-DNP technique and protein immobilization may facilitate the identification of intraligand NOEs in ligand screening for drug discovery. The same method may be applied to in vivo characterization of ligand interactions with cell surface proteins.

中文翻译：

通过超极化放大核超负荷效应，以筛选与固定的靶蛋白结合的配体。

靶蛋白的固定化提高了核弛豫极化转移的交叉松弛速率，但降低了可达到的靶标浓度。通过溶解动态核极化（D-DNP）显示配体自旋超极化可提高观察配体内核Overhauser效应（NOE）的敏感性。这种效应也称为转移的NOE（trNOE），可用于检测结合并获得与结合有关的结构信息。证明了配体4'-羟基偶氮苯-2-羧酸与固定在聚苯乙烯珠上的抗生物素蛋白相互作用的超极化trNOE信号的测量。在包含63.5μM配体和0.83μM可及蛋白结合位点的样品中，D-DNP提供的信号增强作用导致对NOE积累的单次扫描检测，尽管该信号仅在总配体信号的2％处达到峰值。这些堆积曲线允许通过NOE符号的改变和交叉松弛速率的定量确定来确认结合。D-DNP技术和蛋白质固定化的结合可以促进配体筛选中配体内NOE的鉴定，从而发现药物。相同的方法可以应用于体内配体与细胞表面蛋白相互作用的表征。D-DNP技术和蛋白质固定化的结合可以促进配体筛选中配体内NOE的鉴定，从而发现药物。相同的方法可以应用于体内配体与细胞表面蛋白相互作用的表征。D-DNP技术和蛋白质固定化的结合可以促进配体筛选中配体内NOE的鉴定，从而发现药物。相同的方法可以应用于体内配体与细胞表面蛋白相互作用的表征。

更新日期：2020-10-21

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